Vitamin D is one of the most substantial micronutrients, among zinc or vitamin C, with an advantageous influence on patients’ condition in the course of infection-related inflammation of the respiratory tract, with lesser risk and reduced duration of symptoms²⁴. Results of numerous studies from both pediatric and adolescent subjects indicated a critical prevalence of vitamin D deficiency among those infected with SARS-CoV-2. In addition, suggesting that compounds play a preventive role in the severity of that condition^7,25. Our study allowed us to reveal the severity of the vitamin D deficiency within population of infected patients at the time of the COVID-19 pandemic, with further investigation of its role in the infection outcome.

Blood concentration of vitamin D was shown to be associated with season-related changes in UV exposure, higher in summertime versus low levels in winter and autumn²⁶. During the SARS-CoV-2 pandemic, home isolation, reduced outdoor activity, UV exposure blocked by clothes, and the infection itself (often repeatedly occurring) significantly affected vitamin D availability^27,28. We showed that all of our infected patients’ 25(OH)D levels oscillated around values typical for severe deficiency of that compound. And that is no isolated case, as comparable levels were shown even in groups narrowed to asymptomatic/mild asymptomatic individuals^4,29. Those data seemed to be supported by prolonged viral exposure and the fact of commonly occurring deficiencies in Eastern European countries, with around 80% of subjects in Poland around the August/September period³⁰. Additionally, dietary habits with low intake of vitamin D-rich products (fatty fish) further affect low synthesis of that compound^31,32. No influence of sex, age, or even year’s season was demonstrated in reference to the vitamin concentrations during the spread of COVID-19. No significant differences were registered despite slightly lower values in moderate and high-risk infected subjects. Previous studies actually showed that vitamin D levels might influence severity in the infected subjects, but at the same time, they indicated a huge heterogeneity in those observations^11,33. Furthermore, considering other studies, even regular supplementation (with 5000 IU/d) might not be efficient for long-term improvement of vitamin D status in patients and healthy controls, as shown in soccer players during the pandemic²⁷. Similarly, the high single dose of vitamin D3 (200.000 IU) applied in COVID-19-infected patients from the moderate/severe group showed no benefits for hospitalization duration, ICU admission, or mortality^34,35.

Research conducted in the course of the SARS-CoV-2-related crisis suggested significant benefits of higher vitamin D levels and its supplementation in subjects with mild and severe conditions, affecting inter alia hospitalization time and better therapeutic outcomes^10,19,36. In fact, our study revealed that extended hospitalization time was associated with 25(OH)D deficiency and generally higher frequency of patients with values of that compound below the median. Interestingly, another research showed that despite a slightly higher mortality rate, supplementation with vitamin D (25-hydroxyvitamin D) did not affect the hospitalization outcome. Simultaneously, no association was shown between vitamin D levels and the severity of the COVID-19 infection, which is in consent with our results¹⁷. Certain studies indicated better outcomes of the vitamin application in older subjects at higher altitudes, with concomitant speculation that those less severe exclusively might respond effectively³³. Data presented here shed new light on the actual relation between 25(OH)D and SARS-CoV-2 -related clinical aspects. In addition, it might be possible that reported low vitamin D levels result from the infection-related inflammation and not simply deficiency as suggested in previous reports⁹. In accordance, some data suggest that measurement of 25(OH)D (reduced in the course of the inflammatory process) cannot be used for evaluation of actual vitamin D status³⁷.

Substantial part of the project was to demonstrate whether 25(OH)D levels are associated with patients’ conditions and viremia. Meta-analyses demonstrated a significant contribution of low vitamin levels to higher severity and mortality, as well as increased risk of SARS-CoV-2 infection³³. Quite surprisingly, data from our infected subjects aged between 45 and 70 years showed that vitamin D correlated positively with one of the COVID-19 genes expression ORF1ab. Thus, in the patients already infected with the virus, vitamin D concentrations might not have such an unambiguous beneficial effect on the viremia. Noteworthy, approaches for improving the infected patients’ condition through correcting the vitamin D status should also be carefully considered. Extensive meta-analysis showed that only regular daily vitamin supplementation can positively affect the course of the disease, unlike intermittent bolus dosing as that might induce factors inactivating vitamin D (24-hydroxylase and fibroblasts growth factor 23)²².

The same association was reported in the group of patients with high severity and those hospitalized for more than 2 weeks. That could partially explain other studies’ results where some tendencies were observed for even worse hospitalization outcomes, with higher mortality, in patients additionally supplemented with vitamin D¹⁷. In fact, the benefits of vitamin D induction regarding reduced mortality risk were reported exclusively in patients regularly supplemented prior to COVID-19 infection²³. Interestingly, only for ORF1ab any significant correlations were demonstrated, with none in N or E viral gene expression. We presume that lack of clearly substantial associations between 25(OH)D and gene-based viremia, and described above clinical parameters, might result from additional contributing factors. Genetic variants resulting in initially lowered 25-hydroxyvitamin D were the ones with no effect of that compound level on the COVID-19 infection outcome. The same study did not support use of vitamin D supplementation in reducing the risk of infection and therapy outcome¹⁸. On the other hand, in hospitalized patients with standard intervention due to COVID-19, those with implementation of calcifediol showed a lower risk of admission to intensive care units²⁰. Cumulatively, heterogeneity in the results of vitamin D influence on the infection course and outcome indicates need for further investigation of the role of that compound. Presumably, groups of patients with certain stages of inflammatory processes might benefit from vitamin D supplementation, which additionally seems to support a therapeutic approach rather than directly affecting viral biology.

Previous study suggested that intensified supplementation with vitamin D (5000 IU/d versus 1000 IU/d) might improve recovery time for typical symptoms of SARS-CoV-2 infection – cough and ageusia. Those observation, however, were related to patients from mild to moderate severity, with sub-optimal initial concentrations of that vitamin³⁸. Risk analysis revealed no statistically significant influence of 25(OH)D levels on the occurrence of most common symptoms observed in the course of SARS-CoV-2 infection. Therefore, we cannot link any of the well-known symptoms with levels of 25(OH)D in COVID-19-infected patients. Clinical trials with vitamin D supplementation also support our observations, with no differences between COVID-19-infected subjects treated additionally with the vitamin compared to the placebo group in the context of hospitalization duration, admission to ICU, and ventilation requirement³⁹. Despite no relation between vitamin D and infection-related clinical manifestations, other studies suggested the compound’s crucial role in COVID-19 management. In contrast to our subjects with severe deficiency, data from patients with insufficiency/deficiency compared to those with normal levels of around 50 ng/ml showed that vitamin D negatively correlates with mortality rate⁴⁰.

Subsequently, we assessed whether the investigated population of patients demonstrated any relation between the vitamin and changes in laboratory data directly associated with subjects monitoring. First, a positive correlation was found between vitamin D and INR levels. Previous studies demonstrated that, in fact, a low (1000 IU) dosage of that compound only affected INR exclusively compared to 5000 IU/d. In addition, they did not show changes in other hemostasis-related parameters, such as platelets (PLT), APTT, and PT³⁸. With further stratification revealed that that relation is present exclusively in subjects with longer hospitalization duration and those with lower expression of the viral genes. No significant correlations were found here between vitamin D and WBC, leukocyte distribution, or PTL, with similar associations demonstrated in the course of systemic bacterial infection—sepsis (also including PCT, CRP, IL-6 in analysis)⁴¹. One study demonstrated that higher levels of vitamin D applied (5000 IU) resulted in increased blood concentration of D-dimers, with no changes in those with a daily dose of 1000 IU³⁸. Here, only within patients with high expression of COVID-19 diagnostic genes positive association was found between vitamin D3 and D-Dimer levels. At the same time, that subgroup of patients showed a negative correlation between the studied vitamin and hemoglobin concentration.

Considering the diagnostic laboratory data, we further verified the risk of certain occurrence levels in the context of those parameters. We found inter alia that lower vitamin D was associated with achieving lower WBC values in monitoring, with neutrophils being presumably the most affected population. Interestingly, evaluation of small quantities of vitamin D effect on leukocytes showed a decline in lymphocytes predominantly, followed by monocytes, and no changes in neutrophils³⁸. In contrast, the dose of 25 μg calcifediol daily (equivalent of 3000 to 6000 IU/d) led to an increase in lymphocytes and a reduction in neutrophil level, resulting in reduced neutrophil-to-lymphocyte ratio (NLR). Those effects diminished within a maximum 2 months after discharge from the hospital³⁹. In context of affecting leukocytes, vitamin D levels, and supplementation might be more essential as adjunct therapy. To date, it has been found to influence changes in monocytes and other cells significantly, additionally improving steroid activity^42,43,44. Those effects might be linked with reported improvement of the patients’ status and exacerbation reduction, predominantly in asthmatic patients already managed with standard therapy⁴⁵. In reference to the correlation with hemoglobin, we also demonstrated that lower vitamin D could result in lower ferritin concentrations in infected patients. Subjects infected with COVID-19 showed previously that supplementation with low vitamin doses is only associated with significant drop in blood ferritin levels³⁸.

Within last several years, the contribution of vitamin D levels to coagulation system function has been shown in various conditions⁴⁶. Some data suggested that supplementation with calcitriol, in such conditions as cancer, can reduce risk of thromboembolic events⁴⁷. Relation of vitamin D to hemostasis can be linked inter alia to vitamin D receptor (VDR) presence in megakaryocytes—being the source of the platelets⁴⁸. Thus, affecting substantial changes in platelet morphology (including mean platelet volume, MPV) that can influence a higher incidence of thrombosis⁴⁹. Nevertheless, here, we did not find vitamin D levels to contribute to variations in total platelet numbers (PLT) in patients infected with COVID-19. Furthermore, no significant results were obtained when 25(OH)D levels were investigated in the context of their relation to blood saturation, liver function parameters, or coagulation parameters, including INR and D-dimers. It has been recently shown that supplementation with cholecalciferol at high dose (60.000 IU/d for 7 days) can reduce fibrinogen concentration, together with a decline in the frequency of Sars-Cov-2 positive subjects, with no effect on D-dimers²⁹.

This study has several limitations that should be acknowledged. Firstly, the sample size was limited to a single center and a relatively small cohort, which may restrict the generalizability of the findings. Moreover, 25(OH) vitamin D levels were measured only at one time point during hospitalization, which does not allow for assessment of dynamic changes in 25(OH) D status during the course of the illness. Although the expression of the ORF1ab gene was assessed as a marker of active SARS-CoV-2 infection, no viral load quantification was performed, which could have provided additional insights into the relationship between viral activity and immune responses.